ABSTRACT Hematopoietic stem and progenitor cell (HSPC) transplantation (HSCT) is routinely used for the treatment of inborn errors. Ex vivo gene editing/therapy is becoming a successful tool for treatment of patients with bone marrow (BM) failure (BMF) and immunodeficiencies. However, the complete engraftment of HSC in these patients requires larger-than-expected cell doses and HSC transplantation is useful in ameliorating bone diseases like osteogenesis imperfecta. These unexpected observations have not been followed by a stringent mechanistic analysis. Mitochondria are well known metabolic sensors that bridge transcriptional signatures and cellular functions. The mitochondrial content of HSC is elevated but the preferential use of glycolysis and low mitochondrial activity in HSC as supported by a large cohort of experimental data suggest that mitochondrial respiration is more dispensable for HSC than for their cell progeny. Our preliminary data indicate that mitochondrial transfer exists between hematopoietic cells and their surrounding microenvironment with functional consequences on hematopoietic and mesenchymal regeneration after myeloablation. Our data suggests the existence of refined configuration of the mitochondrial fate defining the HSC and microenvironment fate in the regenerating bone marrow by metabolic coupling controlled by two major molecular nodes. We hypothesize that HSPC mitochondria transfer is required for metabolic coupling between HSPC and MSC/P of the BM. The goal of this proposal is to define the mechanisms that control the mitochondrial content and transfer from hematopoietic engrafting cells and their impact on BM mesenchymal regeneration. We will elucidate the mechanisms of mitochondrial transfer in the BM niche and their functional relevance using genetic and pharmacological tools of gain- and loss-of-function and enumeration and functional analysis of biochemical consequences of the traffic of mitochondria in the HSC niche. The mitochondrial transfer reprograms the metabolome of recipient BM MSC/P and this reprogramming is necessary for mesenchymal proliferation and reconstitution of the mesenchymal niche of the BM as well as bone regeneration of the BM after myeloablation. We will determine whether a) the negative regulator role of Cx43 in mitochondrial transfer depends on cell-to-cell contact; b) the mitochondrial transfer from BM HSPC to BM MSC/P induces metabolic reprogramming of the mesenchymal microenvironment resulting in hematopoietic regeneration; and, c) the prevention of AMPK activation is required for BM mesenchymal and hematopoietic regeneration. This proposal will provide light on the molecular basis of hematopoietic-dependent mesenchymal regeneration after transplantation and will identify the role of hematopoietic Cx43 and host AMPK activity on bone marrow metabolic coupling.